Preparing membrane receptors from extracellular baculoviruses

ABSTRACT

Method for producing membrane receptors based on a baculovirus/insect cell system. The invention further includes the receptors produced by the method described herein, a model for studying the properties of these membrane receptors and an assay to screen molecules that are active on these membrane receptors.

FIELD OF THE INVENTION

The invention relates to the production of membrane receptors in abaculovirus/insect cell system.

BACKGROUND OF THE INVENTION

Over the past few years, heterologous expression systems have often beenused to study the expression as well as the pharmacological andbiochemical characteristics of membrane receptors.

Although a significant expression can be obtained in some expressionsystems in mammalian cells, there have been problems, in particular inthe case of some types of receptors such as the G-protein-coupledreceptors.

The G-protein-coupled receptors belong to the superfamily of receptorswith seven transmembrane domains. They comprise, for example, theadrenergic or muscarinic receptors, and all have the same structurewhich is made up of a polypeptide chain comprising seven hydrophobicdomains which cross the membrane lipid bilayer.

When it is desired to express these receptors in mammalian cell systems,a relatively low density of receptors expressed by the said cells,rarely exceeding a few picomoles of receptor per milligram of membraneprotein, is generally obtained. Although these levels of expression aresufficient for a functional and pharmacological characterization, theyclearly limit the type of biochemical, biophysical and structuralstudies which can be carried out. A fortiori, this expression systemcannot be used for the production of receptors in a large quantity, forexample for their therapeutic use.

To increase the quantity of receptors obtained, various teams havesought to produce them in a baculovirus/insect cell system; in manycases, baculoviruses expressing G-protein-coupled receptors have beenable to produce these recombinant receptors in cells of the Spodopterafrugiperda Sf9 or Sf21 lines, up to levels reaching 30 to 100 picomolesper milligram of membrane protein. These systems have made it possibleto make significant progress in the study of the palmitoylation ofreceptors and also to study the effects produced by various agonists andantagonists, or to carry out the reconstitution of artificial receptors.

However, the baculovirus/insect cell system has the major disadvantageof expressing a high proportion of inactive receptors. The receptors,which are recovered in the membrane fraction of the cells infected withthe baculoviruses are in an immature and incompletely glycosylated form.This probably results from a saturation of the normal post-translationalmaturation pathway, which brings about the retention of immaturereceptors in the membranes of the endoplasmic reticulum or in the Golgiapparatus. To obtain functional receptors, it is necessary in this caseto include a purification step based on the biological activity of thereceptor (for example an affinity chromatography step).

It would therefore be necessary to develop a system which makes itpossible to easily separate the plasma membrane comprising the maturereceptors from the other membrane fractions such as the endoplasmicreticulum or the membranes of the Golgi apparatus which comprise thebiologically inactive, immature receptor.

SUMMARY OF THE INVENTION

It has recently been shown that the infection of Sf9 cells with abaculovirus encoding the HIV1 Gag gene (Pr55 Gag) brings about thebudding of particles carrying the Gag protein (Gag particles) which arereleased into the extracellular medium. It has been suggested that theseGag particles carry with them, during their budding, the plasma membraneand the proteins associated with it.

The inventors have formulated the hypothesis that the coexpression, in abaculovirus/insect cell system, of a G-protein-coupled receptor and ofPr55 Gag can promote the release of the Gag particles expressing onlythe mature receptors which are correctly inserted into the plasmamembrane. To test this hypothesis, the inventors infected Sf9 cells withbaculoviruses encoding the human adrenergic receptor β2AR and the Pr55Gag protein. Surprisingly, they then observed that the β2AR receptor isalmost completely absent from the Gag particles, but is, on the otherhand, present at a high density in extracellular baculovirus particles.In addition, the receptors expressed in these extracellularbaculoviruses are correctly glycosylated and normally active.

The present invention relates to the use of these extracellularbaculoviruses for the production of preparations of a membrane receptor.

The subject of the present invention is a method of producing arecombinant membrane receptor in a baculovirus/insect cell system, froma culture of insect cells infected with a recombinant baculovirusexpressing the gene encoding the membrane receptor, which method ischaracterized in that the membrane receptor is obtained fromextracellular baculoviruses produced by the infected cells.

According to a preferred embodiment of the present invention, thereceptor belongs to the superfamily of receptors with seventransmembrane domains; this is for example a receptor of the family ofG-protein-coupled receptors.

Recombinant baculoviruses expressing the gene encoding the membranereceptor which it is desired to produce are obtained by cloning the geneunder transcriptional control of an appropriate promoter of thebaculovirus, according to methods well known per se to persons skilledin the art.

Any strong baculovirus promoter which can be used for the expression ofheterologous genes, such as for example the polyhedrin promoter (polh)or that of the P10 protein, may be used for the production of arecombinant baculovirus which can be used within the framework of thepresent invention.

According to a preferred embodiment of the method in accordance with theinvention, it comprises a step during which the extracellularbaculoviruses produced by the infected cells are harvested and they areseparated from the cellular fractions. The harvesting and the separationof the extracellular baculoviruses may be carried out by successivecentrifugations, for example in the following manner: a firstcentrifugation is carried out at about 500×g, at the end of which thesupernatant containing the extracellular baculoviruses is collected.This supernatant is subjected to a centrifugation at about 45,000×g; theresulting pellet which contains the extracellular baculoviruses isresuspended, and the suspension is subjected to a centrifugation atabout 500×g; the supernatant resulting from this centrifugation iscentrifuged at about 45,000×g, and the pellet, which contains theextracellular baculoviruses, is recovered. Advantageously, theextracellular baculoviruses may also be purified by sucrose gradientcentrifugation, or any other equivalent method.

According to another preferred embodiment of the method in accordancewith the invention, it comprises a step during which the extracellularbaculoviruses produced by the infected cells are lysed; advantageously,it also comprises a step during which the lysate obtained at the end ofthe preceding step is fractionated, and the fraction comprising themembrane receptor is recovered.

The purified preparations and the lysates of extracellularbaculoviruses, as well as their fractions comprising the membranereceptor, which are capable of being obtained by the methods definedabove constitute membrane receptor preparations which also form part ofthe subject of the present invention. These preparations consist ofactive and fully mature receptors, unlike the membrane receptorpreparations obtained in the prior art from the plasma membranes ofinfected cells, which comprise a high proportion of inactive receptors,and which can only be used after an additional step of purification onthe basis of the activity of the relevant receptors, for example afteraffinity chromatography.

By contrast, the membrane receptor preparations in accordance with theinvention are characterized in that, prior to any purification carriedout on the basis of the activity of the relevant receptor, at least 90%,and preferably at least 95%, of the receptor is in an active form.

Preparations, in accordance with the invention, of a membrane receptormay be used to prepare the receptor in a purified form, with a muchbetter yield than that which could be achieved from membrane receptorpreparations obtained in the prior art from the plasma membranes ofinfected cells.

The membrane receptor preparations in accordance with the invention, aswell as the extracellular baculoviruses obtained using the method inaccordance with the invention, may also be used directly, for example asa system for studying the properties of membrane receptors, as a systemfor screening molecules which are active on these membrane receptors, oralternatively for studying their post-translational modifications suchas phosphorylation or palmitoylation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: WAR activity in extracellular recombinant baculoviruses.

FIG. 2: Comparison of the forms of β2AR receptor present in cellmembrane preparations and in extracellular baculoviruses.

FIG. 3: Palmitoylation of β2AR receptors expressed in extracellularbaculoviruses. Clear bars: incorporation in the absence ofisoproterenol; Etched bars: incorporation in the presence ofisoproterenol.

FIG. 4: Comparison of forms of the β2AR receptor. On the y-axis:relative incorporation of ³²P (arbitrary units). On the x-axis:BASAL=control; FRSK=incorporation in the presence of forskolin;cAMP=incorporation in the presence of dibutyril cyclic AMP; andISO=incorporation in the presence of isoproterenol.

FIG. 5: Functionality of the β2AR receptor expressed in extracellularbaculoviruses. On the y-axis: adenyl cyclase activity (in picomoles ofcyclic AMP/min/mg of protein). On the x-axis: BASAL=control;FRSK=incorporation in the presence of forskolin; NaF=incorporation inthe presence of NaF; and ISO=incorporation in the presence ofisoproterenol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be understood more clearly with the aid ofthe additional description which follows, which refers to exemplaryembodiments of the method in accordance with the invention for thepreparation of membrane receptors.

EXAMPLE 1 Preparation of Recombinant Baculoviruses Expressing aG-Protein-Coupled Receptor

A recombinant baculovirus expressing β2AR is obtained by cloning a DNAsequence consisting of the cDNA for β2AR in fusion with the c-mycepitope, obtained as described by MOUILLAC et al. [J. Biol. Chem., 267,21733-21737 (1992)], at the NheI site of the transfection/recombinationvector pJVNheI (marketed by the company INVITROGEN). This vector wastransfected with the linearized genome of a baculovirus AcMNPV (sold bythe company INVITROGEN) into Sf9 cells; the recombinant baculovirusobtained in this manner is called c-myc-β2AR.

In the same manner, the sequence encoding the muscarinic receptor M1 andthe sequence encoding the dopaminergic receptor D1 [respectivelydescribed by ALLARD et al. Nucleic Acid Research, 15, p 10604, (1987)and by DEARRY et al., Nature, 347, p 72, (1990)] were cloned in order toobtain the recombinant baculoviruses (respectively called M1-R and D1-R)expressing these receptors.

a) Culture and infection of the cells, and harvesting of theextracellular baculoviruses:

Sf9 cells are cultured at 27° C. in culture flasks in a 100 mlsuspension (BELLCO GLASS) in supplemented GRACE medium (GIBCO)containing 10% foetal calf serum (FCS), and 0.001% pluronic acid. 60 mlof suspension of cells (2×10⁶/ml) are infected with the recombinantbaculovirus expressing P2AR, D1 or M1, at a multiplicity of infectionvarying between 2 and 5.

The cells are harvested by centrifugation at 500×g for 5 min at 4° C.

The viral particles are isolated after harvesting the cells, bycentrifugation of the culture supernatant at 45,000×g for 20 min at 4°C. The pellets obtained are resuspended at 4° C. in a volume ofphosphate-buffered saline (PBS) equal to {fraction (1/10)}th of thevolume of the initial culture, and centrifuged at 500×g for 5 minutes at4° C.; the supernatant for this centrifugation at 500×g is againcentrifuged at 45,000×g for 20 min at 4° C.

b) Purification of the baculovirus particles on a sucrose gradient

The pellet of viral particles which is obtained from 100 ml of culturesof Sf9 cells infected with the recombinant baculovirus expressing β2AR,M1 or D1 is resuspended in 1.2 ml of TE solution (10 mM Tris-HCl, 1 mMEDTA, pH 7.4), in the presence of protease inhibitors).

The suspension is deposited at the top of a tube containing a lineargradient (25%-56%) of sucrose in TE solution. The tubes are centrifugedat 100,000×g for 90 minutes. The gradient is collected from the top tothe bottom of the tube, in 20 fractions. The first fraction has a volumeof 1.4 ml, and the other 19 are 500 μl.

EXAMPLE 2 Demonstration of the β2AR Activity in the ExtracellularRecombinant Baculoviruses

The Sf9 cells infected with the recombinant baculovirus expressing β2ARare cultured, and the baculoviruses are harvested as described inExample 1 a), 24 hours, 48 hours, 72 hours, 96 hours and 120 hours afterthe infection.

The activity of the β2AR receptors is evaluated by measurements ofsaturation and of competitive binding which are carried out as describedby BOUVIER et al. [Mol. Pharmacol. 33 :133-139 (1982)] using[¹²⁵I]iodocyanopindolol ([¹²⁵I]ICYP) as labelled ligand.

The pellets of viral particles which are obtained are resuspended at 4°C. in the buffer which will be used for the reaction. Aliquots of thissuspension of viral particles, corresponding to 0.2 to 1 μg of proteinsare mixed with 5 to 350 pM of radioligand [¹²⁵I]ICYP in a final volumeof 500 μl. The non-specific binding is evaluated using 10 μM ofalprenolol.

Under these conditions, the β2AR activity is detected in the cellculture supernatants from 48 hours after the infection, reaches itsmaximum 72 hours after the infection, and remains constant up to 120hours after the infection.

These results are illustrated in FIG. 1.

This figure also shows the results obtained, under the same experimentalconditions, on culture supernatants of cells infected both with therecombinant baculovirus c-myc-β2AR, and a recombinant baculovirusexpressing the HIV Gag protein (=β2AR; ♦=β2AR+Gag). It is observedthat, contrary to what was initially expected, the presence of the Gagprotein does not increase the quantity of β2AR in the culturesupernatants.

It is also observed that the β2AR activity detected in the supernatantsdoes not come from the cell lysis, since this activity appears 48 hoursafter the infection; that is to say at a time where the majority of theinfected cells are still viable, and does not increase between 72 and120 hours after the infection, in spite of the substantial cell lysiswhich occurs at this time.

The nature of the particles in the supernatant carrying the β2ARactivity was checked by electron microscopy after labelling theseparticles with the aid of an antibody directed against the c-mycantigen, or an antibody directed against the β2AR receptor. It was thusobserved that the particles recognized by either of these antibodies are15×100 nm rods, which corresponds to extracellular baculoviruses.

In the case of the co-infection with a baculovirus expressing the Gagprotein, the presence of particles exhibiting the morphology of the Gagparticles, and which are recognized by an anti-Gag antibody, isobserved, in addition, in the supernatant; however, unlike theextracellular baculoviruses, these Gag particles are only very weaklyrecognized by the anti-c-myc and antiβ2AR antibodies.

The presence of the β2AR receptor was also verified in the preparationsof recombinant baculovirus c-myc-β2AR which are purified on a sucrosegradient, as described in Example 1 b) above.

The β2AR activity was determined according to the protocol described inExample 2 above, on various fractions of the gradient.

In parallel, the detection of the vp80, gp67 and vp39 antigens of thebaculovirus AcMNPV, using a polyclonal antibody directed against theseantigens, was carried out on the same fractions. The results obtainedshow that the β2AR activity cosediments with the viral particles.

All the results obtained above show that not only are molecules of thereceptor expressed in the recombinant extracellular baculoviruses, butalso that they are active molecules.

The quantification of the β2AR activity in the recombinant extracellularbaculovirus preparations purified on a sucrose gradient makes itpossible to evaluate the density of the active receptor at about 25pmol/mg of total proteins.

EXAMPLE 3 Comparison of the Forms of the β2AR Receptor Which are Presentin Cell Membrane Preparations and in the Extracellular Baculoviruses

Sf9 cells infected with the recombinant baculovirus c-myc-β2AR areharvested 72 hours after the infection. The extracellular baculovirusesc-myc-β2AR are harvested from the culture supernatant of these cells,and the viral particles are purified as described in Example 1b).

The membranes of the Sf9 cells are prepared as follows: the cells arecentrifuged at 500×g for 5 minutes at 4° C., rinsed once with PBS bufferat 4° C., and resuspended in lysis buffer (20 mM Tris-HCl, 5 mM EDTA, pH7.4 containing 5 μg/ml leupeptin, 5 μg/ml of trypsin inhibitor and 10μg/ml of benzamidine) at 4° C. The cells are then lysed by sonication,the lysates are centrifuged for 5 min at 500×g at 4° C. and thesupernatants centrifuged at 45,000×g for 20 min at 4° C. The pellets areresuspended at 4° C. in reaction buffer (75 mM Tris-HCl (pH 7.4), 12.5mM magnesium chloride, 2 mM EDTA), in the presence of proteaseinhibitors.

6 mg of the cell membrane preparation or of the purified baculoviruspreparation are added to 5 ml of solubilization buffer (10 mM Tris-HCl,100 mM NaCl, 2 mM EDTA, pH 7.4, 0.3% n-dodecyl maltoside (BOEHRINGERMANNHEIM) in the presence of protease inhibitors. The solubilization iscarried out for 90 min at 4° C.

The solubilized receptors are purified by affinity chromatography asdescribed below. The ALPRENOLOL-SEPHAROSE affinity matrix is synthesizedaccording to the method of BENOVIC et al. [J. Biol. Chem., 262:9026-9032, (1987)]. This matrix is used to purify c-myc-β2AR accordingto the protocol described by MOUILLAC et al. [J. Biol. Chem., 267:21733-21737, (1992)]. All the buffers comprise n-dodecyl maltoside(0.05%).

The preparations obtained after affinity chromatography are concentratedusing CENTRIPREP and CENTRICON cartridges (AMICON)and the quantity ofc-myc-β2AR in each sample is determined using [¹²⁵I]-iodocyanopindolol([¹²⁵I]ICYP) as described by MOUILLAC et al. [J. Biol. Chem., 267:21733-21737, (1992)]. The preparations of viral particles, of membranesor of β2AR purified by affinity chromatography are subjected to apolyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE),under non-reducing conditions, on 10% gel plates. The proteins separatedon the gels are transferred onto nitrocellulose and revealed with ananti-c-myc mouse monoclonal antibody, and a second anti-mouse antibodycoupled to alkaline phosphatase or to horseradish peroxidase. Theresults are illustrated by FIG. 2.

The Western blotting of the cell membrane preparation (FIG. 2, lane 1)shows the presence of several immunoreactive bands, between 40 and 50kDa.

The Western blotting of the preparations of β2AR purified by affinitychromatography (FIG. 2, lane 2) shows a single and broad immunoreactiveband, between 46 and 50 kDa, which represents the biologically activemature form of the β2AR receptor.

The Western blotting of the purified extracellular baculoviruspreparation (FIG. 2, lane 3) also shows the presence of a single andbroad immunoreactive band between 46 and 50 kDa.

These results show that the β2AR receptor molecules present in theextracellular baculoviruses represent only the biologically active form,unlike the β2AR receptor molecules present in the cell membranepreparations, which represent a mixture of active and inactive forms.

EXAMPLE 4 Pharmacological Properties of Various Receptors Expressed inthe Extracellular Baculoviruses

Preparations of extracellular baculoviruses expressing the β2AR, M1 orD1 receptors are obtained as described in Example 1 above.

The binding of each of the receptors to the ligand is evaluated asdescribed in Example 2 above.

The competitive binding trials in the presence of agonists are carriedout using 70 pM of [¹²⁵I]ICYP as radioligand. The concentration of thenon-labelled ligand varies from 10⁻⁴ to 10⁻¹² M.

The saturation assays of the M1-muscarinic (M1-R) and D1-dopaminergic(D1-R) receptors expressed in the viral particles are carried out using1-100 nM [3H]pirenzepine (NEN, DUPONT) and 0.02-3 nM[¹²⁵I]-R(+)SCH-23390 (NEN, DUPONT) with 5-10 μg or 1-2 μg of protein forM1-R and D1-R respectively. To evaluate non-specific binding, 1 μMatropine (RBI) is added to the reaction mixture for M1-R, and 10 μMhaloperidol (RBI) for D1-R.

The results of these experiments are illustrated by Table I below.

TABLE I BMax Kd pmol/mg of Ki Receptor Ligand pM protein μM β2AR [¹²⁵I]ICYP 49.4 ± 11.5 Epinephrine 8.98 ± 4.02 Arterenol 3.27 ± 0.38 M1 [³H]-Pirenzepine 1360 ± 670  5.56 ± 0.46 D1 [¹²⁵I] -SCH23390 118 ± 63  5.21± 0.84

These results show that various receptors of the G-protein-coupledreceptor family are expressed in an active form in extracellularbaculoviruses.

EXAMPLE 5 Palmitoylation of the β2AR Receptor Expressed in ExtracellularBaculoviruses

The viral particles expressing c-myc-β2AR are prepared as described inExample 1 above, and the pellet resuspended in PBS. 1 mCi of[³H]palmitate dissolved in dimethyl sulphoxide is added to the viralparticles. The reaction is carried out for defined periods in thepresence or the absence of 1 μM (final concentration) of isoproterenol.

The results are illustrated by FIG. 3:

Legend to FIG. 3:

□: incorporation in the absence of isoproterenol;

□: incorporation in the presence of isoproterenol.

EXAMPLE 6 Comparison of the Forms of the β2AR Receptor

The viral particles expressing c-myc-β2AR are prepared as described inExample 1 above, and the pellet resuspended in a buffer (100 mMTris-HCl, 10 mM MgCl₂, pH 7.4 and protease inhibitors). 1 volume ofextracellular baculoviruses and 1 volume of phosphorylation mixture (2.3μCi/pl of [γ³²P]ATP, 10 mM Tris-HCl, 2 mM MgCl₂, pH 7.4, 25 mMphosphoenol pyruvate, 0.3 mM GTP, 1 mM ATP, 4 U/ml of pyruvate kinaseand 20 U/ml of myokinase) are mixed. The reaction is carried out for 25min at 30° C. At the end of the reaction, the incorporation of ³²P ismeasured in the absence of activator (control) or in the presence of 1μM of isoproterenol, or of 100 μM of dibutyril cyclic AMP, or of 100 μMof forskolin. The results are illustrated by FIG. 4.

Legend to FIG. 4:

On the y-axis: relative incorporation of ³²P (arbitrary units)

On the x-axis:

BASAL: control

FRSK: incorporation in the presence of forskolin

cAMP: incorporation in the presence of dibutyril cyclic AMP

ISO: incorporation in the presence of isoproterenol

EXAMPLE 7 Functionality of the β2AR Receptor Expressed in ExtracellularBaculoviruses

The viral particles expressing c-myc-β2AR are prepared as described inExample 1 above, and the pellet resuspended in a buffer (75 mM Tris-HCl,12.5 mM MgCl₂, 2 mM EDTA, pH 7.4 and protease inhibitors). 20 μl ofextracellular baculovirus suspension are mixed with 30 μl of reactionmedium containing 0.2 mM ATP, 0.090 mM GTP, 0.20 mM cAMP, 0.20 mMisobutylmethylxanthine, 1 μCi [γ³²P]ATP, 5 mM phosphoenol pyruvate, 0.3U of pyruvate kinase and 2 U of myokinase. After incubating for 30 minat 37° C., the reactions are stopped by the addition of 1 ml of stopsolution (0.4 mM ATP, 0.3 mM cyclic AMP and 25,000 cpm of tritiatedcyclic AMP). The activity was determined in the absence of activator(control) or in the presence of one of the following activators: 1 μM ofisoproterenol, 10 μM NaF or 100 μM of forskolin. The results areexpressed in picomoles of cyclic AMP produced per minute and permilligram of protein. These results are illustrated by FIG. 5.

Legent to FIG. 5:

On the y-axis: adenyl cyclase activity (in picomoles of cyclicAMP/min/mg of protein)

On the x-axis:

BASAL: control

FRSK: incorporation in the presence of forskolin

NaF: incorporation in the presence of NaF

ISO: incorporation in the presence of isoproterenol

These results show that the β2AR receptor present in the extracellularbaculoviruses is in an environment which reproduces the natural membraneenvironment, and that the extracellular baculovirus preparations cantherefore be used in all membrane receptor applications where areproduction of this environment is desirable.

What is claimed is:
 1. Isolated baculovirus viral particles obtained bya method of producing a recombinant membrane receptor proteincomprising: introducing a baculovirus expression vector encoding saidmembrane receptor protein into an insect host cell; culturing theresultant insect host cell for a sufficient time to permit expression ofsaid membrane receptor protein and baculovirus viral particles;separating the cells from the baculovirus viral particles; and isolatingthe baculovirus viral particles.
 2. The recombinant baculovirus viralparticles of claim 1, wherein said membrane receptor protein belongs tothe superfamily of receptors having seven transmembrane domains.
 3. Therecombinant baculovirus viral particles of claim 2, wherein saidmembrane receptor protein belongs to the family of G-protein-coupledreceptors.
 4. The recombinant baculovirus viral particles of claim 4,wherein at least 90% of said membrane receptor protein is in activeform.
 5. The recombinant baculovirus viral particles of claim 1, whereinat least 95% of said membrane receptor protein is in active form.